Lactobacillus johnsonii BS15  for 28 days. From days 22 to 28, the mice in the CW and PW groups were subjected to water-avoidance stress (WAS). The issue of whether psychological stress–induced memory dysfunction can be prevented via L. johnsonii BS15 pretreatment to modulate the gut–brain axis was investigated. Results show that L. johnsonii BS15 enhanced gut development by increasing villus height in the jejunum and ileum as well as villus height:crypt depth ratio in the ileum. L. johnsonii BS15 increased the activities of digestive enzymes, including trypsin and lipase in the jejunum and ileum. The intestinal goblet cell number was also increased by L. johnsonii BS15 pretreatment. Moreover, L. johnsonii BS15 balanced the gut microbiota by increasing the log10 DNA gene copies of Lactobacillus spp. and L. johnsonii and decreasing that of Enterobacteriaceae in the cecum. L. johnsonii BS15 also exerted preventive effects on intestinal permeability WAS by modulating diamine oxidase and D-lactate levels in the serum and mRNA expression levels of the tight junction proteins claudin-1, occludin, and ZO-1 in the jejunum and ileum. L. johnsonii BS15 pretreatment modulated inflammatory factors, specifically tumor necrosis factor-alpha, interferon-gamma, and interleukin-10. L. johnsonii BS15 pretreatment improved their performance in two behavioral tests, namely the novel object and T-maze tests. This result indicates that psychological stress–induced memory dysfunction possibly could be prevented through the gut–brain axis. In addition, L. johnsonii BS15 exerted beneficial effects on the hippocampus by modulating memory-related functional proteins, especially those related to synaptic plasticity, such as brain-derived neurotrophic factor and stem cell factor. Moreover, L. johnsonii BS15 recovered antioxidant capacity and exerted protective effects on mitochondrion-mediated apoptosis in the hippocampus. Collectively, the modulation of the gut–brain axis by L. johnsonii BS15 could be considered a promising non-invasive treatment modality for psychological stress–induced memory dysfunction.Researchers are attempting to harness the advantages of the gut–brain axis to prevent neurocognitive disorders by enhancing intestinal health. In this study, four groups of ICR mice were orally gavaged with either phosphate-buffered saline (control and CW groups) or the probiotic strain  Psychological stress is common in modern society and has been observed to decrease the functioning of human physiological systems . Stress Several studies that adopted human and rodent models report that the intestinal environment can modulate cognitive behaviors . The conLactobacillus rhamnosus R0011 and L. helveticus R0052) ameliorates colitis and stress-induced colonic dysfunction in rodents and prevents memory dysfunction induced by acute psychological stress in mice. L. rhamnosus R0011 and L. helveticus R0052 in water for 7 days improves barrier function in the intestines of rats to prevent bacterial translocation against chronic psychological stress. In addition, L. rhamnosus JB-1 can regulate emotional behaviors and the expression of the central γ-aminobutyric acid receptor in the hippocampus of mice  to test whether psychological stress–induced memory dysfunction can be prevented through the gut–brain axis in mice. L. johnsonii BS15 was isolated from homemade yogurt collected from Hongyuan Prairie, Aba Autonomous Prefecture, China. In a previous study, we found that L. johnsonii BS15 exerts beneficial effects by attenuating the hepatic inflammatory reaction and mitochondrial injury, thereby balancing the gut environment and preventing non-alcoholic fatty liver disease  under an anaerobic environment for 36 h at 37°C. Heterotrophic plate counting was performed to determine the amounts of bacterial cells. Bacterial cells were collected and washed with saline. Afterward, the bacterial cells were suspended in phosphate-buffered saline (PBS) at pH 7.0. The concentration of suspension was set to 1 × 109L. johnsonii BS15 CFU/mL. In a previous work, we confirmed that a daily amount of 0.2 mL solution with 1 × 109L. johnsonii BS15 CFU/mL  is the optimal dose  for 28 days. The mice in the control and P groups were weighed  on the morning of day 28, and feed intake was also recorded. The average daily feed intake (ADFI), average daily gain (ADG), and feed conversion ratio (FCR) were calculated. All mice were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle. All animal experiments were performed following the guidelines for the care and use of laboratory animals approved by the institutional ethical committee .ICR male mice  were provided by Dashuo Biological Institute . The animals were weighed  and then divided into four groups. Each group contained six cages with seven mice per cage. The animals were provided with a normal chow diet purchased from Dashuo Biological Institute . After stabilizing for 7 days, the four groups were weighed (starting weight) and then orally gavaged with either PBS (pH 7.0) (control and CW groups) or The first day after 1-week stabilization was defined as day 1. From days 22 to 28, the mice in the CW and PW groups were subjected to WAS. The mice were placed on a small platform surrounded by shallow water at room temperature for 60 min each day. Food and water were deprived during the WAS test. The mice in the control group and P groups were prevented from consuming food and water during the WAS test.TM RT reagent kit with gDNA Eraser . The cDNA products were stored at −20°C until subsequent tests. In a subset of experiments, 8–10 mice from each group were selected for the T-maze test. The habituation and training phases of the T-maze test were performed on day 21 and lasted for 6 days until day 27. On day 28, the testing phase of the T-maze test was conducted after the WAS test for the CW and PW groups. Another 10–12 mice from each group were randomly selected for the novel object test. The novel object test was performed on day 28 after the mice in the CW and PW groups were subjected to the WAS test. All mice that underwent behavioral tests were not selected during sample collection to avoid the carryover effects of behavioral testing on other biomarkers, such as inflammation markers  were evaluated by weighing the heart, liver, spleen, kidneys, pancreas, stomach, and adipose tissues  of the mice in the control and P groups. Parts of two small intestinal sections  of the mice in the control and P groups were fixed in 4% formalin solution to measure villus morphology and observe goblet cells. Blood was collected from the mice in all groups by cardiac puncture, and the blood samples were immediately placed in ice and centrifuged. The isolated serum was frozen at −20°C along with fecal samples collected to quantify microbial content until further analysis. A small sample of the hippocampus and the epithelial tissues of the jejunum and ileum were removed and washed with ice-cold sterilized saline. Subsequently, these samples were frozen in liquid nitrogen and then stored at −80°C. The other parts of the hippocampus, jejunum, and ileum were separately removed and frozen at −20°C until further analysis. After the sampling, the samples stored at −80°C were removed, and the RNA of the hippocampus, jejunum, and ileum were extracted using the EZNA total RNA kit  following the manufacturer’s guidelines. Total RNA (1 μg) was synthesized into first-strand complementary DNA (cDNA) by using a PrimeScript markers . Two obsThe novel object test was performed to assess memory using the method of In brief, the mice that did not undergo the WAS test were placed in a dark open-field arena  and allowed to freely explore for 1 h for habituation. Two different objects, namely blue and orange (#2) tube caps of the same size and shape and a smooth pebble of proper size , were exposed to the mice after the WAS test or habituation. Behavioral assessment consisted of familiarization and testing phases.The mice were placed in opposite corners of the arena where objects #1 and #2 were placed in advance. The mice were allowed to explore both objects freely for 5 min. The objects were then removed, and the mice were allowed to rest for 20 min before the testing phase.∗ hereafter) and object #3. Memory was evaluated as the frequency of exploring object #3 in contrast to object # 1∗ during the testing phase. An exploration ratio represented the proportion of smelling bouts related to the new object versus the old one (frequency of smelling #3/(frequency of smelling #1∗ + frequency of smelling #3). A ratio of 0.5 represented impaired hippocampus-dependent memory as no discrimination was observed between the two objects. Exploration in the present test was defined as orienting toward the object with the nose pointing toward the object within 1–2 cm.Object #2 was replaced by object #3 while the mice were resting. After the rest period, the mice were re-exposed to object #1  and two goal arms . Owing to the natural tendency of mice to explore a novel environment, they were first placed at the base of the start arm and freely allowed to enter one of the goal arms. In the second trial, the mice were observed to choose the other goal arm that had not been visited. This behavior could reflect memory of the first choice. Alternation is very sensitive to memory dysfunction, especially that related to the hippocampus, and represents a model of working memory .In the present study, a rewarded T-maze test was chosen over a spontaneous T-maze test because mice can run more trials each day before getting sated . A mixtuDuring the habituation phase (3 days), the mice were softly stroked and slowly picked up and put down three times each day (3 min each time) to ensure that they became accustomed to touch by the operators. In addition, given that rodents are wary of eating anything new, the mice were fed with 0.5 ml of the food reward each day to familiarize them with its taste. The training phase (4 days) was performed after habituation. In brief, the mice were placed in the T-maze with their arms open and allowed to explore freely for 10 min. The mice in the start arm were then allowed to run toward one of the goal arms. One of the goal arms was provided with food in a well, whereas the door of the other goal arm was blocked. No more than 3 min of training time was given until the mice definitively learned that the well was empty. Each mouse was trained four times each day .n = 10).Each mouse was tested for 4 days with five trials per day. The test was started at the same time (10:00 a.m.) every day. The mice were allowed to explore the whole maze before reaching satiation. The WAS test was initiated before the test started each day as needed. With one of the goal arms blocked , the mice from the start arm were allowed to run toward the open goal arm and consume the reward therein. They were immediately returned to the start arm as soon as they learned that the well was empty, and the operator opened the door of the blocked goal arm. With 0 s  or 1 min  of retention interval, the mice were allowed to run again from the start arm and choose one goal arm. The mice were allowed to consume the reward if correct; if incorrect, they were removed after they definitively discovered that the sample well was empty. Hippocampus-related working memory with 0 s and 1 min retention intervals was separately assessed as the ratio of correct times to total trial times . Villus height (Vh) and crypt depth (Cd) of the jejunum and ileum were measured using an image analysis program , and the Vh/Cd ratio was calculated. The sections were dewaxed in xylene, rehydrated through a graded ethanol series, and washed with distilled water. The sections were then stained with Alcian Blue-periodic acid-Schiff (PAS) to observe goblet cells. The goblet cells were stained with PAS and examined under a light microscope . The goblet cells were measured using image analysis software . Six slices were selected from each group, and the number of goblet cells per view in each group was counted and averaged for statistical analysis.D–lactate content; diamine oxidase (DAO) activity; and expression of Bcl-2 and Bax, two apoptosis-related proteins in the hippocampus, in the serum were quantified using ELISA kits specific for mice  following the manufacturer’s instructions. Amylase, trypsin, and lipase activities of the jejunal and ileal contents, including antioxidant indexes in the hippocampus, were measured using commercial kits . These antioxidant indexes included total antioxidative capacity (T-AOC); catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity; and malondialdehyde (MDA) and GSH contents. Moreover, the contents of inflammatory factors, including interleukin (IL)-1β, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), in the jejunum and ileum were determined via ELISA by using reagent kits specific for mice .The ΔΔCt method was applied to estimate mRNA abundance. The samples (n = 6) in each group were analyzed in triplicate. Ct was calculated as (Cttarget – Ctβ –actin)treatment – (Cttarget – Ctβ –actin)control. β-actin was used as the eukaryotic housekeeping gene to normalize relative gene-expression levels. Mean values of the measurements were used to calculate mRNA-expression levels of the N-methyl-D-aspartate receptor (NMDAR), cyclic amp response element binding protein (CREB), brain-derived neurotrophic factor (BDNF), and c-Fos. The mRNA expression levels of the stem cell factor (SCF), neural cell adhesion molecule (NCAM), Bcl-2, Bax, Bad, Bcl-xL, caspase-3, and caspase-9 in the hippocampus were measured. The mRNA-expression levels of IL-1β, TNF-α, IFN-γ, IL-4, IL-10, IL-6, claudin-1, occludin, and zoluna occludens protein-1 in the jejunum and ileum were also determined.The prepared cDNA products from the hippocampus, jejunum, and ileum were used for polymerase chain reaction (PCR). The test was performed using a CFX96 real-time PCR detection system  with iTaq universal SYBR green supermix . The protocols for thermocycling were as follows: 5 min at 95°C, 40 cycles of 10 s denaturation at 95°C, and 30 s annealing/extension at optimum temperature , 2. The Lactobacillus spp., Enterobacteriaceae, and L. johnsonii were estimated in the cecum following the method of TM real-time system  and SYBR® Premix Ex Taq II  were used to perform qPCR. ® Premix Ex TaqTM II (12.5 μL), forward and reverse primers (1 μL), sterile deionized water (9.5 μL), and DNA template (1 μL). PCR was performed as follows: 95°C for 1 min, 40 cycles of 94°C for 15 min, and annealing at optimal temperatures for 30 s at 72°C. The specificity of the PCR primers was regulated by generating melting curves.The populations of total bacteria, Bacteroidetes, Firmicutes, P < 0.05 were considered statistically significant.The data on growth performance were analyzed on a per-cage basis. All other data were analyzed on the basis of the individual mouse. Statistical analysis was performed using one-way ANOVA, followed by Duncan’s multiple-range test for multiple comparisons . Differences at P = 0.276) and starting weight (P = 0.251) was found between the control and P groups. However, the mice in the P group had significantly higher (P < 0.05) final weight, DWG (P < 0.01), and FCR than those in the control group. However, pretreatment with L. johnsonii BS15 did not improve ADFI (P = 0.669). Moreover, no significant difference was observed (P > 0.05) in all determined tissue weight indexes between the two experimental groups.The results of growth performance and tissue weight indexes are shown in L. johnsonii BS15 significantly improved Vh in both the jejunum (P < 0.05) and ileum (P < 0.01)  in the P group than in the control group. However, the Vh/Cd ratio did not significantly change (P = 0.217) in the jejunum  and ileum (P < 0.01) of the mice in the P group significantly increased. However, no difference in amylase activity was detected  between the two groups. Moreover, the number of goblet cells in the jejunum and ileum significantly increased (P < 0.001) in the P group compared with that in the control group (Pretreatment with  < 0.01) . However jejunum . The resol group , 4.Lactobacillus spp. (P < 0.05) and L. johnsonii (P < 0.01) significantly increased, whereas those of Enterobacteriaceae significantly decreased (P < 0.01) in the P group compared with that in the control group. The populations of Lactobacillus spp. (P < 0.05) and L. johnsonii (P < 0.001) significantly increased, whereas those of Enterobacteriaceae (P < 0.01) decreased in the PW group compared with those in the CW group. However, the population of total bacteria (P = 0.479), Firmicutes (P = 0.273), and Bacteroidetes (P = 0.987) was not significantly different among all experimental groups. The WAS test did not alter the microbial populations because no significant changes were observed between the control and CW groups (P > 0.05).Microbial populations in the cecum were quantified via qPCR. The results are presented in D-lactate in the serum were significantly higher (P < 0.01) in the CW group than in the control group. WAS also significantly decreased the mRNA-expression levels of the three tight junction proteins claudin-1, occludin, and ZO-1 in the jejunum and ileum of the mice in the CW and control groups. Without WAS, L. johnsonii BS15 pretreatment did not significantly influenced the contents of DAO (P = 0.923) and D-lactate (P = 0.947) in the serum. Moreover, the contents of D-lactate in the serum of the mice in the CW and PW groups were not significantly different (P = 0.053). However, L. johnsonii BS15 pretreatment significantly altered (P < 0.05) the contents of DAO in the serum of the mice in the CW and PW groups. Regardless of whether WAS was initiated or not, the mRNA-expression levels of the three tight junction proteins in the jejunum and ileum were significantly higher (P < 0.001) in the mice pretreated with L. johnsonii BS15 than in the mice without probiotic gavage.The results of intestinal permeability are presented in P < 0.01), TNF-α (P < 0.01), and IFN-γ (P < 0.001) between the control and CW groups. Moreover, WAS significantly decreased the mRNA expression level of IL-10 (P < 0.05) in the CW group compared with that in the control group. Except for IL-1β , the same changes caused by WAS were detected (P < 0.01) in the protein contents of TNF-α, IFN-γ, and IL-10  the protein contents and mRNA expression levels of IL-1β, IL-4, and IL-6 between the control and P groups and between the CW and PW groups. The absence of WAS did not significantly change (P > 0.05) the protein contents between the control and P groups. Furthermore, the mRNA expression levels of all inflammatory factors did not change  in the P group than in the other groups. However, the mRNA-expression levels and protein contents of TNF-α and IFN-γ were significantly lower (P < 0.01) in the PW group than in the CW group. The mRNA-expression levels and protein contents of IL-10 were also significantly higher (P < 0.01) in the PW group than in the CW group.The results of protein contents and mRNA expression levels of inflammatory factors in the ileum are shown in nd IL-10 . HoweverP = 0.492) among all groups in terms of the correct time with 0 s of retention interval (P < 0.01) the correct time with 1 min of retention interval between the control and P groups (P < 0.05) in the PW group than in the CW group, but the difference between the control and P groups was not significant (P = 0.465). Moreover, WAS decreased the exploration ratio (P < 0.001) in the novel object test, whereas L. johnsonii BS15 pretreatment significantly increased (P < 0.05) the exploration ratio in the PW group compared with that in the CW group (L. johnsonii BS15 pretreatment did not significantly change (P = 0.400) the exploration ratio between the control and P groups.interval . HoweverP groups . In addiCW group . HoweverP < 0.001) in the P group compared with the control group. In addition, the mRNA-expression levels of BDNF (P < 0.05), CREB (P < 0.001), SCF (P < 0.05), and NMDAR (P < 0.001) were significantly higher in the P group than in the control group. However, the mRNA-expression level of c-Fos did not significantly change (P = 0.942) between the two groups. Moreover, L. johnsonii BS15 pretreatment significantly increased the mRNA-expression levels of BDNF, CREB, SCF, c-Fos, and NMDAR against WAS; their mRNA-expression levels in the PW group were significantly higher than those in the CW group . However, the mRNA expression levels of NCAM in the four experimental groups were not evidently different (P = 0.608).P = 0.125). Moreover, no significant difference in any of the indexes related to antioxidant capacity between the control and P groups was found (P > 0.05). However, WAS significantly influenced antioxidant capacity; the activities of T-AOC, SOD, CAT, and GSH-Px were lower and the level of MDA was higher in the CW group than in the control group . Furthermore, the activities of T-AOC, SOD, GSH-Px, and CAT were higher in the PW group than in the CW group . In addition, the level of MDA was lower (P < 0.05) in the PW group than in the CW group.The evaluation results of antioxidant capacity in the hippocampus are presented in P > 0.05) in any of the indexes was noted between the control and P groups. However, Bax content and the mRNA-expression levels of Bax and caspase-3 were significantly higher in the CW group than in the control group  and the PW group . bcl-2 content and the mRNA-expression levels of bcl-2 and bcl-xl were also lower in the CW group than in the control group  and the PW group . In addition, the mRNA-expression levels of bad (P = 0.408) and caspase-9 (P = 0.543) in the hippocampus among the four groups were not significantly different.The evaluation results of apoptosis-related functional protein contents and mRNA-expression levels in the hippocampus are presented in L. johnsonii BS15 pretreatment exerted beneficial effects by increasing the Vh and Vh/Cd ratio, indicating that pretreatment improved development of the jejunum and ileum. As a consequence of improved intestinal development, the activities of different digestive enzymes can also be improved by the commercial probiotic L. plantarum, which enhances growth performance   . In the athogens . In the L. johnsonii BS15 pretreatment. Moreover, the mRNA-expression level of IL-1β was upregulated by WAS. Impairments of the intestinal barrier may be associated with an increase in proinflammatory cytokines in the ileum because the mRNA-expression levels and most protein contents of TNF-α, IFN-γ, and IL-1β substantially increased. Furthermore, three important proinflammatory cytokines were detected in the CW group, and the level of IL-10, which is secreted as an anti-inflammatory cytokine, decreased. The increase in proinflammatory cytokines was also detected by L. johnsonii BS15 pretreatment can reduce levels of TNF-α, IFN-γ, and IL-1β in the small intestines of mice, especially under WAS. This result indicates the ability of L. johnsonii BS15 to protect the intestines from inflammation. This finding is in agreement with that of Bifidobacteria infantis 35624, to rats. An intestinal barrier with damaged integrity was observed by L. johnsonii BS15 may exert beneficial effects on maintaining intestinal integrity. Notably, psychological stress negatively affected gut integrity and intestinal inflammation without damaging the gut microbiota. High populations of Lactobacillus spp. and L. johnsonii and low populations of Enterobacteriaceae in the cecum were detected in the groups pretreated with L. johnsonii BS15 before (P vs. control group) and after (PW vs. CW group) applying psychological stress. Meanwhile, psychological stress did not alter the microbial populations because no significant changes were observed between the control and CW groups. These results indicate that L. johnsonii BS15 possibly protected the mice from psychological stress by primarily improving their intestinal health  and not by maintaining gut microbiota.The relationship between changes in gut microbiota and intestinal inflammation is complex and does not appear to reflect a simple cause. Probiotics may prevent intestinal inflammation by altering the composition of the microbial community in the intestines . Reid etL. johnsonii BS15 pretreatment effectively prevented the negative changes induced by WAS. Moreover, no difference was observed in the T-maze tests with a 0-s retention interval. However, the damage caused by WAS and the preventive effects exerted by L. johnsonii BS15 were both demonstrated when the retention interval was increased from 0 s to 1 min, indicating that 1 h of WAS for 7 days could produce only mild cognitive impairment, a condition that can only be revealed by increasing cognitive demands .HW, JX, NS, and XN performed the experiments. HW, YS, and YB analyzed and interpreted the results. YS, HW, TZ, DZ, and YB drafted and revised the manuscript. All authors read and approved the final manuscript and contributed to the design of the experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the pathology-oriented study of depression, inflammation hypothesis has received increasing attention for recent years. To mimic the depressive state caused by inflammation, rodents injected intraperitoneally with lipopolysaccharide (LPS) are usually used to stimulate an immune response. However, the dose of LPS that causes depressive-like behavior varies widely across many literatures. Previous study has uncovered the non-linearity in the dose-effect relationship for the depressive-like behavior induced by LPS administration, while the reason for this is still unclear. The present study aims to investigate the underlying mechanisms of this non-linear dose-dependent relationship. Four groups of mice were injected intraperitoneally with different doses of LPS . The tail suspension test was conducted to evaluate the depressive-like behavior within 23–25 h after the LPS administration. The neuroplasticity was assessed by the levels of related proteins, TrkB and PSD-95, and by the quantification of neurons using Nissl staining. The levels of the two metabolites of the kynurenine (KYN) pathway, 3-hydroxykynurenine (3-HK) and kynurenic acid (KYNA), in the brain were analyzed by LC-MS/MS. Activation of microglia and astrocytes in the brain were also determined by immunohistochemistry and western blotting, respectively. The results showed that, compared with the control group, the mice in the 0.8 mg/kg LPS-treated group exhibited a remarkable increase of immobility time in the tail suspension test. The neuroplasticity of mice in the 0.8 mg/kg LPS-treated group was also significantly reduced. The neurotoxic metabolite, 3-HK, was accumulated significantly in the hippocampus of the 0.8 mg/kg LPS-treated mice. Surprisingly, the 2 mg/kg LPS-treated mice did not exhibit a remarkable change of 3-HK but expressed increased KYNA significantly, which is neuroprotective. Furthermore, the activation of microglia and astrocytes, which were recognized as the primary source of 3-HK and KYNA, respectively, corresponded to the content of these two metabolites of the KYN pathway in each group. Consequently, it was speculated that the homeostasis of different glial cells could lead to a non-linear dose-dependent behavior by regulating the KYN pathway in the LPS-induced depressive-like mice. Depression is a prevalent psychiatric illness, afflicting patients physically and mentally . By the Lipopolysaccharide (LPS) is a potent activator of the immune system . IntrapeIn fact, the pathophysiology of depression is associated with dysfunctions of many systems in the body, including the immune system, monoaminergic system, and glutaminergic system. One potential intersection point of these three systems is the kynurenine (KYN) pathway, of which neuroactive products are involved in the interface between the immune response and serotoninergic neurotransmission via catabolism of tryptophan (Trp) to KYN, ultimately altering downstream synaptic glutamate neurotransmission . FurtherIn the brain, KYN can be distinctively processed by either microglia or astrocytes to produce different neuroactive compounds, such as 3-hydroxykynurenine (3-HK) and kynurenic acid (KYNA). Basically, KYNA can be formed mainly in astrocytes , which iTherefore, we hypothesized that the non-linearity in the dose-effect relationship for the depressive-like behavior induced by intraperitoneal injection (i.p.) of LPS might be due to modification of downstream metabolites of the KYN pathway caused by the homeostasis fluctuations of astrocytes and microglia, which led to the further changes in the CNS. The present study was aimed to evaluate the effect of different doses of LPS on depressive-like behaviors, neuroplasticity, the immune system, and KYN pathway in the brain of mice.p < 0.05). Unpredictably, the immobility time of 2 mg/kg LPS-treated mice in the TST was only slightly increased, without statistical significance compared to the control group. The effects of different doses of LPS on the depressive-like behavior of mice were assessed by the immobility time in the tail suspension test (TST). As shown in To examine whether the LPS-induced non-linear dose-dependent behavior was associated with neuroplasticity, the status of neurons in the hippocampus A and theGiven the regulatory role of the KYN pathway in the brain development and immune response, the metabolite levels of the KYN pathway in the brain were detected via LC-MS/MS analysis . The resβ, TNFα, TGFβ, and IL-10 mRNA levels in the brain of mice were detected by RT-PCR (p < 0.01). The mRNA levels of IL-1β, IL-10, and TGFβ mRNA in the hippocampus of mice treated with different doses of LPS were similar to the changes of that of TNFα. The mice exposed to 2 mg/kg LPS expressed significant increased IL-1β and TGFβ mRNA levels in the cortex (p < 0.01), while the 0.32 mg/kg and 0.8 mg/kg LPS-treated group showed a slight increase of these two mRNA levels in the cortex. Not consistent with the behavioral results, these cytokine mRNA levels in the hippocampus and cortex were mostly dose-dependent.Many in vivo studies examining the impact of LPS use the endpoint measure of cytokine expression. In our study, the cytokine IL-1y RT-PCR . The resp < 0.05), whereas the other two LPS-treated groups showed no statistical significance. The status of astrocytes was detected by glial fibrillary acidic protein (GFAP) using western blotting, and the results of GFAP showed a dose-dependent activation of astrocytes  to clear glutamate spillover in the CNS . Althougtivation , which ativation . In addition, there are some limitations in this study that could be addressed in future research. First, CD68/ED1 can be used as the microglia marker to investigate the status of activated microglia. Second, cell and animal experiments with related inhibitors need to be conducted to further elucidate the causal role of homeostasis imbalance of microglia and astrocytes in the alterations of metabolites of the KYN pathway in LPS-induced depressive-like mice. Taken together, our present study proposed for the first time that LPS administration could change the levels of the metabolites of the KYN pathway by regulating the homeostasis of different glial cells, and ultimately affect the neuroplasticity of the central nervous system. This study attested to the claims that LPS administration could induce non-linear dose-dependent behavioral changes and was the first to explain this non-linear dose-effect relationship from the alterations of neuroplasticity and inflammatory response. Nevertheless, this work was expected to provide a basis for the establishment of a much more reliable depression model induced by LPS and a powerful physiological basis for the study of inflammation-associated depression.Male C57BL/6J mice (7–8 weeks old) were purchased from the Beijing Weitong Lihua Experimental Animal Technology Company. Procedure rooms were maintained at a temperature of 23 ± 1 °C and a humidity of 60% ± 5%. All animals were bred and housed four per cage with standard food and water available ad libitum. They were maintained on a 12 h light/dark cycle (lights on at 08:30 a.m.). All animal care and experimental protocols were approved by the Committee on Animal Care and Use of the Institution of Medical Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College .Escherichia coli (serotype O55:B5) was purchased from Sigma-Aldrich and freshly dissolved in sterile saline prior to injection. 5-HT, 5-HIAA, TRP, KYNA, 3-HK, and 4-acetamidophenol  were obtained from Sigma-Aldrich . Acetonitrile and formic acid (mass-spectrometry grade) were provided by Fisher Co., Ltd. . Trifluoroacetic acid (TFA) was purchased from Dikma .Lipopolysaccharide (LPS) from n = 12) by body weight: control group , 0.32 mg/kg LPS-treated group, 0.8 mg/kg LPS-treated group, and 2 mg/kg LPS-treated group. The tail suspension test (TST) was conducted within 23–25 h after the i.p. injection of LPS. After the TST, the mice were sacrificed by decapitation. The cerebral cortex and hippocampus from nine mice randomly selected in each group were rapidly separated and stored at −80 °C before further analysis. The remaining three mice in each group were perfused transcardially with saline, followed by cold 4% paraformaldehyde (PFA) in PBS. The brains were excised and fixed in 4% PFA for 24 h. Following paraffin embedding, serial 4 μm-thick coronal sections were performed with Nissl or immunohistochemistry (IHC) staining by standard protocols.After 7-day acclimation, the mice were randomly divided into four groups  and it was then transferred onto a nitrocellulose membrane. After a 2 h of block in 7.5% milk solution, the membrane was incubated with primary antibodies at 4 °C overnight, including anti-TrkB , anti-GFAP  and anti-PSD-95 , respectively. Subsequently, the membrane was rinsed three times (10min per time) and then incubated with appropriate horseradish peroxidase-linked secondary antibodies  at room temperature for another 2 h. Finally, the protein was detected by enhanced chemiluminescence. The grey intensity values of bands were analyzed using Image J 1.46r software .The proteins of the hippocampus or cortex were extracted using a proper amount of protein extraction solution containing 1% protease inhibitors and 1% phosphatase inhibitors. Following centrifuging at 20,000 β forward 5′-CACTACAGGCTCCGATGAACAAC-3′, IL-1β reverse 5′-TGTCGTTGCTTGGTTCTCCTTGTAC-3′, TNFα forward 5′-ATGTCTCAGCCTCTTCTCATTC-3′ and TNFα reverse 5′-GCTTGTCACTCGAATTTTGAGA-3′, IL-10 forward 5′-TCCCTGGGTGAGAAGCTGAAGAC-3′, IL-10 reverse 5′-CACCTGCTCCACTGCCTTGC-3′, TGFβ forward 5′-ACCGCAACAACGCCATCTATGAG-3′ and TGFβ reverse 5′-GGCACTGCTTCCCGAATGTCTG-3′. Forty cycles of the profile were run. Amplicon size and specificity were confirmed by the melting curve analysis. Relative mRNA expression of the target gene was defined as 2−ΔΔCt.Total RNA was extracted from the cortex using TRIzol regent according to the manufacturer’s instructions . Trace contamination of DNA was removed by DNase digestion. The Reverse Transcription System (Roche) was used for the first strand cDNA synthesis. Quantitation of all gene transcripts was performed in IQ 5.0 ABI 7500  using Top Green qPCR SuperMix  The sequences of primers were as follows: IL-1Nissl staining was carried out as described before . Brain s2O2 for 25 min. Then they were blocked for non-specific antigen binding using 3% BSA for 30 min at room temperature, which was followed by an overnight incubation with primary antibody against Iba1  at 4 °C. Sections were then rinsed in 0.1 M PBS for 3 times and incubated with horseradish peroxidase-linked anti-mouse IgG for 50 min at room temperature. One set with hematoxylin counterstaining was used for cellular location, which was performed according to the manufacturer’s instructions . Sections were then rinsed, dehydrated in 75%, 85%, and 100% ethyl alcohol, cleared in xylene and coverslipped with Permount  in a fume hood.IHC analysis was conducted as Zhu et al. described before with a slight modification . BrieflyThe images of sections were acquired at 20× (for Nissl staining) or 40× (for IHC) magnifications utilizing the Aperio CS2 system . With a threshold that discriminated staining from the background best, the integrated optical density (IOD) above the threshold was calculated for each image with equal size by ImageJ software .p < 0.05. Calculations were performed using SPSS statistical software (version 17.0) .Data were expressed as mean ± SEM. One-way ANOVA followed by post hoc LSD test was performed to determine the significance for multiple comparisons when the data meet normal distribution. When the data did not meet the requirements of normal distribution, Mann–Whitney U test was conducted. Significance was set at


To achieve CHIP overexpression in animal models, we intravenously injected mice with AAV/BBB, a new serotype of adeno-associated virus that features an enhanced capacity to cross the blood-brain barrier. We also generated gene knock-in mice that overexpressed CHIP in neural tissue. Our results demonstrated that CHIP overexpression in mice suppressed 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage, including movement impairments, motor coordination, and spontaneous locomotor activity, as well as loss of dopaminergic neurons. In vitro and in vivo experiments showed that overexpression of CHIP inhibited the pathological increase in Drp1 observed in the PD models, suggesting that CHIP regulates Drp1 degradation to attenuate MPP+/MPTP-induced injury. We conclude that CHIP plays a protective role in MPP+/MPTP-induced PD models. Our experiments further revealed that CHIP maintains the integrity of mitochondria.Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson’s disease (PD). Carboxyl terminus of Hsp70-interacting protein (CHIP) is a key regulator of mitochondrial dynamics, and mutations in CHIP or deficits in its expression have been associated with various neurological diseases. This study explores the protective role of CHIP in cells and murine PD models. In SH-SY5Y cell line, overexpression of CHIP improved the cell viability and increased the ATP levels upon treatment with 1-methyl-4-phenylpyridinium (MPP Parkinson’s disease (PD), which is characterized by clinical features such as bradykinesia, tremor, rigidity, and autonomic dysfunction, is the second most common neurodegenerative disease . Progresin vivo and in vitro PD models shows that modulation of mitochondrial dynamics can attenuate PD-associated impairments. Thus, a potential avenue of PD treatment involves preserving the normal function of mitochondria [Mitochondrial dysfunction is considered the core factor in the pathogenesis of PD. Mitochondria are highly dynamic organelles essential for energy conversion, and the high energy demand of neurons renders them vulnerable to mitochondrial dysfunction . Indeed,chondria .Pink1-mutated Drosophila ameliorated locomotion impairments, loss of dopaminergic neurons, and mitochondrial defects [The carboxyl terminus of Hsp70-interacting protein (CHIP) is a co-chaperone protein essential for protein quality control. In addition, CHIP is considered to be a central molecular player in mitochondrial stress signaling and cellular homeostasis. CHIP dysfunction has been implicated in various neurological diseases . CHIP al defects . Further defects .+), which damages the mitochondrial respiratory chain in dopaminergic neurons and eventually leads to cell death [+-treated human neuroblastoma cells have been used as cellular models of PD in vitro. To explore the protective role of CHIP in PD models, we used adeno-associated virus (AAV) to upregulate the expression of CHIP in adult mice. MPTP was then injected to induce PD-like symptoms. AAV/BBB is a new variant of AAV that can cross the blood-brain barrier with high efficiency, enabling effective intravenous gene transfer to the mouse central nervous system. We also generated a gene knock-in mouse model based on Nestin-cre/loxP-driven CHIP overexpression that allows for the selective induction of CHIP in neural tissue. We then studied the effect of CHIP overexpression in vivo on the amelioration of motor impairments, cell viability, and mitochondrial homeostasis in MPTP-induced PD models. Our results may provide new evidence for the protective effect of CHIP in PD and other neurodegenerative diseases.1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been widely used to generate models of PD. After crossing the blood-brain barrier, MPTP is metabolized to 1-methyl-4-phenylpyridinium  leakage. After treatment with 2 mM of MPP+ for 24 h, we observed an obvious cell viability decrease and extracellular LDH increase. Overexpression of wild-type CHIP prior to MPP+ exposure increased cell viability and decreased LDH levels relative to the vector control (+ exposure and found the cells to be significantly more sensitive to MPP+-induced injury (Human neuroblastoma (SH-SY5Y) cells were treated with different concentrations of MPPl damage . Westernl damage –1E. To d control , 2B. We d injury , 2B.+ exposure resulted in enhanced ATP levels, while reduced ATP production was observed in cells transfected with CHIP-shRNA, compared to the ATP levels in controls with endogenous CHIP levels  staining and analysis of the mitochondrial form factors. Greater numbers of mitochondrial fragments were detected in CHIP-knockdown cells, while in CHIP-overexpressing cells, the extent of mitochondrial fragmentation was less severe than in cells receiving only MPP+ , 4B.STUB1 gene to achieve neural CHIP expression. NES-CHIP mice and wild-type littermates aged 10–12 weeks were chosen for the experiment. The distribution and expression of proteins were assessed with CHIP/GFP double staining in the AAV group and with CHIP immunofluorescence in the knock-in group. Compared with the mice that received vehicle, mice that received AAV/BBB-CHIP-GFP or AAV/BBB-GFP showed increased expression of GFP. However, CHIP was significantly upregulated only in the AAV/BBB-CHIP-GFP group. Similar results were confirmed in the NES-CHIP mice  is the marker of dopaminergic neurons, and the abundance of TH-positive cells in the SNpc and striatum can reflect the severity of neuronal loss after MPTP exposure. Immunohistochemistry results showed that CHIP protected TH-positive cells from MPTP damage. Cell counting confirmed a statistical difference –8D. Accoth day after the last injection of MPTP. The SNpc and striatum were isolated and total proteins were extracted. The five targets assessed by western blot included the following fusion- and fission-associated proteins: Opa1, Mfn1, Mfn2, Drp1, and Fis1. Seven days after the end of MPTP administration, Drp1 levels in the AAV/BBB-GFP or vehicle pre-treated mice were significantly higher than those in the AAV/BBB-CHIP-GFP pre-treated mice  prevented this rising trend in Drp1 level. Moreover, the results of MitoTracker Red staining showed less mitochondrion fragmentation after MPP+ treatment in the CHIP overexpression group.Drp1 is involved in the pathogenesis of PD. In an  on Drp1 . Drp1 al release . ApplicaD models , 38. OurRecombinant adeno-associated viruses are useful vectors in research, as they do not integrate into the host genome. AAV/BBB is a new serotype of AAV that can cross the blood-brain barrier with enhanced efficiency, and is based on the work by Gradinaru et al. on AAV-PHP.B, which is a capsid variant of AAV9 that can transfer intravenously administered genes throughout the CNS with greater efficiency than the current serotype . Though +-induced damage in SH-SY5Y cells and ameliorate the motor function of MPTP-treated mice by regulating Drp1, a molecule functionally downstream from CHIP. Further studies are required to clarify the interaction between CHIP and Drp1 as well as the role of Drp1 in the pathogenesis of PD. In addition, our results support further investigation of gene therapy for neurodegenerative diseases, including PD.In this study, we demonstrated that overexpression of CHIP could prevent MPP2. To obtain the in vitro experimental PD model, 0.5, 1, 2, and 3 mM of MPP+  were added to different wells, which were then cultured for 24 h to determine the optimal concentration of MPP+.The human neuroblastoma cell line SH-SY5Y was purchased from the American Type Culture Collection . Cells were used and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and cultured at 37° C in a moist atmosphere containing 5% CO5 cells) using LipofectamineTM 2000  to induce overexpression of human CHIP. To generate the CHIP knockdown and control groups, cells were transfected with an shRNA (5’-GGCCTTGTGCTACCTGAAGAT-3’) against human CHIP and a scrambled control shRNA with no significant homology to any known gene sequence, respectively. The plasmid and shRNA were made by GenePharma . After 6 h of incubation, the medium was replaced with regular culture medium, MPP+ was added, and the cells were cultured for an additional 24 h.SH-SY5Y cells were transfected with the CHIP-WT plasmid  dissolved in DMSO to an SH-SY5Y cell suspension 6 h after transfection, when the MPPAfter the completion of the indicated experiments, Cell Counting Kit 8  was added to each well and incubated for 1 h at 37° C. The optical density at 450 nm was then measured using a microplate reader to index cell viability. Each experiment was repeated thrice.An LDH cytotoxicity assay kit  was used to assay the amount of LDH released from damaged cells. Absorbance at 490 nm was used to quantify the amount of LDH. The ratio of LDH activity in the supernatant to total LDH activity was used to index cell death as outlined in the manufacturer’s instructions.The level of ATP in SH-SY5Y cell lines under different conditions was determined with an ATP bioluminescence assay kit . ATP concentration was determined by phosphorylating glycerol, resulting in a colorimetric product with an absorption at 570 nm, which is proportional to the amount of ATP present.SH-SY5Y cells were loaded with preheated, 100 nM MitoTracker Red CMXRos  prepared with complete medium. After incubation at 37° C for 30 min, the culture medium was replaced with fresh medium and the cells were live-imaged with the Leica Multiphoton system.This study was approved by the Life Science Ethics Review Committee of Zhengzhou University.CHIP-with-floxed-STOP-codon mice were generated with CRISPR/Cas9 . The CHIP knock-in and wild-type mice both had a C57BL/6 genetic background. Mice with the CHIP-with-floxed-STOP codon were crossed with Nestin-cre mice  to yield NES-CHIP animals. All the animals were housed in a specific-pathogen–free environment with a 12/12-h light/dark cycle and free access to water and food. The temperature and humidity were controlled at 22 ± 1° C and 60 ± 5%, respectively. Only male mice were used in the experiments.Movement disorders were assessed using the pole test. The apparatus was a wooden pole with a height of 60 cm and a width of 1 cm, with a 2-cm diameter wooden ball affixed to the top. The pole was covered in gauze to prevent sliding. Each mouse was placed on the wooden ball, and the total time taken for the mouse to climb down the pole was measured. Pre-training was necessary to ensure that the mice attempted to move down the pole after they were placed on the top.Motor coordination and learning ability were evaluated using an accelerating rotarod test. The mouse was placed on an automatic rotarod bar with a diameter of 3 cm, and the apparatus then accelerated from 4 to 40 rpm within 5 min. Each test comprised three trials with an interval of 1 h between trials. The latency to falling from the rod was recorded. Before drug treatment, mice were trained for three consecutive days to ensure that they could remain on the rod for 5 min at baseline.Spontaneous locomotor activity was analyzed using an open-field test. The apparatus consisted of a square arena and surrounding walls, and measured 40 × 40 × 40 cm. The floor was divided into 16 squares. Each mouse was placed in the central square of the arena, and the number of squares that the mouse passed over within 5 min was recorded. The arena was wiped with alcohol after each test.st, 3rd, 5th, and 7th days after MPTP injection.AAV/BBB-CHIP-GFP and the control AAV/BBB-GFP were made by Hanbio Biotechnology . When the male wild-type mice reached 6–8 weeks of age, 100 μL of AAV were injected into the tail vein. At 10–12 weeks of age, the AAV-injected mice, the NES-CHIP mice, and the wild-type mice received four intraperitoneal injections of 20 mg/kg of MPTP  or 0.9% phosphate-buffered saline (PBS) at 2-h intervals. Our experiment thus featured five groups of animals: AAV/BBB-CHIP-GFP, AAV/BBB-GFP, vehicle (PBS), NES-CHIP, and wild-type. 8 to 10 mice were included in each group. Behavioral testing was conducted at baseline and on the 1Mice were sacrificed under anesthesia at day 7 following the last injection. For immunohistochemistry, the mice were perfused successively with 1 × PBS and 4% paraformaldehyde , and the whole brain was removed by craniotomy. The mice brains were post-fixed in 4% PFA overnight and dehydrated with 20% and 30% sucrose solution at 4° C. Coronal slices were cut at a thickness of 20 μm with a freezing microtome  and stored at -20° C. For immunoblotting, the brains were separated into different regions and frozen at -80° C.2O2, and nonspecific sites were blocked with 5% normal sheep serum in 0.3% triton X-100. The slices were incubated overnight at 4° C with the primary antibody, anti-tyrosine hydroxylase . On the following day, the slices were incubated with secondary antibody at room temperature. Diaminobenzidine (DAB) was used to visualize antibody binding. For immunofluorescence, the slices were incubated with the primary anti-CHIP  and anti-GFP antibodies  under the aforementioned conditions. The secondary antibodies were tagged with different fluorescent labels. Cell nuclei were stained with DAPI .The frozen brain slices were returned to room temperature and washed thrice in PBS to remove the tissue freezing medium, and antigens were retrieved. A SPlink Detection Kit was used for immunohistochemistry . Endogenous peroxidase was blocked by incubation with 0.3% HTotal protein was extracted from brain tissue or cultured cells with ice-cold RIPA buffer and protease inhibitor mixture (Solarbio). Equal amounts of protein per track were loaded onto 10% SDS-PAGE gels  for electrophoresis and then transferred onto PVDF membranes (Millipore) at low temperature. After blocking in 5% non-fat milk, the membranes were incubated overnight with primary antibody followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. The primary antibodies included anti-GAPDH , anti-beta tubulin , anti-TH , anti-CHIP , anti-Opa1 , anti-Drp1 , anti-Mfn1 , anti-Mfn2 , and anti-Fis1 . Membranes were developed using the ECL luminescence reagent  and the protein bands visualized by enhanced chemiluminescence. Signal intensities were quantified with Image J software  and normalized to an internal control protein.t-tests were used to compare two groups, and one-way analysis of variance (ANOVA) was used for comparisons of multiple groups. The level of statistical significance was set to P < 0.05.The data are expressed as mean ± standard deviation (SD) and analyzed with SPSS21.0 statistical software . Student’s


In this work, we developed a deep learning platform for de novo design of putative inhibitors of SARS-CoV-2 main protease (Mpro). Our methodology consists of 3 main steps: (1) training and validation of general chemistry-based generative model; (2) fine-tuning of the generative model for the chemical space of SARS-CoV- Mpro inhibitors and (3) training of a classifier for bioactivity prediction using transfer learning. The fine-tuned chemical model generated > 90% valid, diverse and novel (not present on the training set) structures. The generated molecules showed a good overlap with Mpro chemical space, displaying similar physicochemical properties and chemical structures. In addition, novel scaffolds were also generated, showing the potential to explore new chemical series. The classification model outperformed the baseline area under the precision-recall curve, showing it can be used for prediction. In addition, the model also outperformed the freely available model Chemprop on an external test set of fragments screened against SARS-CoV-2 Mpro, showing its potential to identify putative antivirals to tackle the COVID-19 pandemic. Finally, among the top-20 predicted hits, we identified nine hits via molecular docking displaying binding poses and interactions similar to experimentally validated inhibitors.The global pandemic of coronavirus disease (COVID-19) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) created a rush to discover drug candidates. Despite the efforts, so far no vaccine or drug has been approved for treatment. Artificial intelligence offers solutions that could accelerate the discovery and optimization of new antivirals, especially in the current scenario dominated by the scarcity of compounds active against SARS-CoV-2. The main protease (M The global pandemic of coronavirus disease COVID-19) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) created a rush to discover drug candidates against the virus 9 caused . As of JCoronaviridae [Coronaviruses are enveloped, single-stranded RNA viruses members of the family aviridae . Their gaviridae –12. ORF1aviridae –15. In Saviridae .pro or 3C-like) of SARS-CoV-2 is a cysteine protease and consists of a homodimer organized in three domains (I–III) [pro is conserved among coronaviruses, sharing ~ 76% sequence similarity with SAR-CoV-1 Mpro, and there are no homologs in humans, making it an attractive target for drug discovery [pro suggests that previously described inhibitors could be used as templates to design new inhibitors to boost the drug arsenal against SARS-CoV-2.The main protease 3-chymotrypsin-like (M (I–III) . The act (I–III) . Mpro isiscovery . FurtherThe development of treatments against SARS-CoV-2 is a fast-changing field and a comprehensive review is out of the scope of this work. However, we instruct the interested reader to a recent review from our group about COVID-19 molecular targets and drug repurposing strategies –22. Due pro); and a classification model to predict the bioactivity of the generated molecules on SARS-CoV-2 Mpro. The molecules predicted as active were further analysed using molecular docking to investigate possible interactions with Mpro.In this work we used ULMFiT  to trainSimplified Molecular Input Line Entry Specification) strings [L = {H, C, N, O, P, S, Br, I, Cl, F}. We also removed molecules with less than 10 or more than 50 heavy atoms. The filtering and standardization steps were implemented using RDKit 2020.01.1 (https://www.rdkit.org/).We used ChEMBL 26  and PubC strings . We downpro available on PubChem (AID: 1706). Originally, AID1706 consisted of 405 active molecules, but we augmented it with 224 inhibitors collected from literature by Tang et al., (https://github.com/tbwxmu/2019-nCov) [For fine-tuning, the dataset consisted of over 280 K molecules screened against SARS-CoV-1 M19-nCov) . In totaIn this work, we used molecules represented as SMILES strings as input to the model Fig. . Each SMIn order to use molecules as SMILES strings as input, the SMILES were initially split into individual characters or tokens, representing the individual atoms in the molecule and special chemical environments, . After tokenization, we used a string-to-integer dictionary to convert each token to a unique integer. In total, the dictionary consisted of N entries, including beginning of string (BOS), end of string (EOS) to represent the start and end of each SMILES string, respectively. We also added padding tokens (needed for the classification task) and UNK tokens to deal with tokens that were not covered by the dictionary, which could be useful when dealing with molecules with exotic groups. To summarize, each molecule was represented by an integer array, where each number represented an atom or chemical environment.We used AWD-LSTM as a base architecture , which iWe initially trained the model using the default 1152 hidden units of ULMFit. However, the total training time was superior to the GPU time available. Therefore, we changed the number of hidden units to 512 while still maintaining performance. During training, the embedding layer receives the inputs and maps them to a latent feature space that contains the contextualized information about a molecule, which can be learned. The embedding and LSTM layers are the encoder of the model, responsible for learning the “chemical language” and short and long-term dependencies between each token. The final layer was the decoder and consisted of a linear layer with a softmax activation that outputs probabilities for each predicted token.For fine-tuning the classifier, we used the same AWD-LSTM architecture as the language model but augmented it with two additional linear blocks with relu activations. In other words, only the linear blocks of the classifier were trained from scratch, which makes the ULMFit approach very flexible in the quantitative structure–activity relationship (QSAR) context, since the chemical language learned by the general model can be reused .ht of the encoder concatenated with the max-pooled and mean-pooled activations of previous time steps. This pooling operation returns the maximum and average activations of previous time steps, allowing the model to focus on the most important features to make a prediction [The input to the classifier is the activation of the last time step ediction . In addi–3. We randomly selected 10% of the data as a validation set to monitor the performance and avoid overfitting. For fine-tuning, we started with the pretrained model and fine-tuned it using discriminative learning rates and gradual unfreezing as proposed by Howard & Ruder in the original ULMFit paper [layer−1 = ηlayer/2.6, used in the original ULMFiT approach, where η is the learning rate and layer is the number of a specific layer.We trained the general chemical model from scratch for 10 epochs using a constant learning rate of 3 × 10it paper . In thisit paper , it is sit paper . The leaTraining with gradual unfreeze initially trains only the linear blocks of the classifier, while keeping the parameters of the encoder frozen. We initially trained the classifier for 4 epochs and then unfroze and fine-tuned each layer every 3 epochs until convergence and all layers were fine-tuned . This mehttps://docs.fast.ai). The codes and models for reproducibility are freely available on request. All codes were written in Python 3 and ran on Google Colaboratory (Colab)  using Ubuntu 17.10 64 bits, with 2.3 GHz cores and e 13 GB RAM, equipped with NVIDIA Tesla K80 GPU with 12 GB RAM.We implemented our model using Fastai v1 library  . The sampling temperature is a hyperparameter that adjusts the output probabilities for the predicted tokens and controls the degree of randomness of the generated SMILES and the confidence of predicting the next token in a sequence . Lower td as Eq. :1\documeyi is the softmax output, T is the temperature and j ranges from i to K number of maximum tokens to sample from the model.where pro using X-ray crystallography was used as an external evaluation set (https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem/Downloads.html). Since the dataset was highly unbalanced, we used the area under the precision-recall curve (AUC-PR) as the key metric to evaluate the performance, which is more informative in this scenario [The classifier performance was evaluated with fivefold cross-validation. We performed two types of splitting: (1) random split into training, validation and test sets using a 80:10:10 ratio, and (2) Scaffold-based splitting in order to ensure that the same scaffolds were not present in training and validation sets. In addition, a dataset of 880 fragments screened against SARS-CoV-2 Mscenario . The AUCwhere TP, TN, FP and FN are the numbers of true positives, true negatives, false positives and false negatives, respectively.http://chemprop.csail.mit.edu/predict) [We also compared the performance of our classifier on the external test set with Chemprop, a freely available message passing neural network (MPNN) that has been used to repurpose drugs to SARS-CoV-2 (predict) .We evaluated the chemical space coverage by computing Uniform Manifold Approximation and Projection (UMAP) plots of Morgan circular fingerprints or Extended Connectivity Fingerprints (ECFP) of length 1,024 bits and radius of 2 bonds. UMAP is a dimensionality reduction method used to visualize high-dimensional data (in this case ECFP4 fingerprints) in just 2 dimensions (2D). Using this method, similar molecules are clustered close to each other, while also preserving the global structure of the original high-dimensional data . In addipro active site. The molecules were scored with the ChemPLP scoring function, using the default search speed parameter of PLANTS v1.2 (search speed = 1). All docking calculations were carried out using the crystal structure of SAR-CoV-2 Mpro . Before submitting our molecule library to docking, we performed a round of redocking in order to validate our protocol. The results are shown in the Additional file A molecular docking simulation was carried out using the Protein–Ligand Ant System (PLANTS) v.1.2 docking software. The active site Cys145 was used as the center of the simulation box and 15Å were added to each cartesian direction, in order to include the S1/1′, S2 and S3 subsites of MIn order to prioritize compounds for purchase and synthesis, we submitted the top 20 predicted hits to a pan assay interference compounds (PAINS) filter to remove potentially problematic molecules from our final selection. All calculations were carried out on the FAF-DRUGS4 server .We initially validated the chemical model trained on ChEMBL to access its potential to generate molecules using SMILES strings. The main metrics have been used to validate generative models in other works , 40, 46.We initially investigated the performance of different sampling temperatures on the proportion of valid, unique and novel molecules generated. Our results are summarized in Additional file T = 0.8, we obtained a good compromise of validity (98.73 ± 0.15%), uniqueness (98.69 ± 0.17%) and novelty (86.57 ± 0.36%) scores . The top-10 most common scaffolds were relatively simple, fragment-sized, with less than 30 heavy atoms and consisting of at most two 6-membered rings Fig. . In addiIn terms of novelty, 3291 (43.66%) of the scaffolds were not present on the training data. In general, the frequencies of each novel scaffold in the generated set were low; each representing only 0.03% of all scaffolds Fig. . StructuThe UMAP shows the scaffolds of 2,000 randomly selected molecules from ChEMBL and the generated set . As expected, with T = 0.80 and the maximum size of SMILES set to 50, in order to generate molecules in the drug-like chemical space.We also investigated the distribution of molecular weights as a function of the maximum size of the SMILES strings the model could generate. Figure pro inhibitors . The generated molecules displayed a slight shift to lower values of molecular weight (MW), logP and number of heavy atoms, indicating that, in general, the model generated smaller and more hydrophilic molecules compared to MWe also investigated the ease of synthesis of the generated molecules. For this analysis, we used the synthetic accessibility score (SAS), which penalizes molecules with highly complex structures, such as high number of stereocenters and multiple ring systems. The SAS score ranges from 1 to 10, with high values being assigned to more complex and difficult to synthesize molecules . The gepro inhibitors training set, showing that the transfer learning process did not simply copy molecules from the training data. As shown on the UMAP plots, the generated molecules not only share the chemical space with Mpro inhibitors but they extend it by filling gaps with novel molecules, corroborating our previous finding about the similar physicochemical parameters  of novel molecules compared to Mpro inhibitors. The novel scaffold also filled gaps in chemical space demonstrating that the fine-tuned model successfully approximated the target chemical space  chemical space, which is an interesting feature for de novo design; if the model could generate novel scaffolds it might be possible to find scaffold hopping opportunities and new chemical series. Among the 70,000 generated molecules, we found 35,713 unique scaffolds 52.89%); of which 35,538 (99.51%) were novel compared to the training set. The UMAP plot shows the overlap in chemical space between Bemis-Murcko scaffolds of the generated molecules and M9%; of whWe also analyzed how different the novel scaffolds were from the training set scaffolds. Overall, the novel scaffolds were structurally different to their closest neighbor in the training set, with a Tanimoto coefficient of 0.420 ± 0.10. Some novel scaffolds displayed small modifications compared to their closest neighbors, such as the insertion or removal of a few atoms between rings, the substitution of oxygen for sulfur atoms and substitution of one ring Fig. a. In gencreativity, in a sense that it introduced modifications to existing scaffolds and generated novel structures. This creativity can also be seen in other works. For instance, the RXRs and PPARs inhibitors generated by Merk et al. showed a similar biphenyl scaffold and most modifications were on the radicals. Although the authors did not make the training set available, it is possible that the biphenyl moiety was present on the training set [In general, the model showed some ning set . In anotning set . A recenning set .The works summarized above are a small sample of what is possible with modern generative models, showing how different deep learning strategies can be used to generate novel scaffolds with a range of modifications. However, it is important to highlight that the true impact of such modifications in terms of intellectual property and publication quality is still an open question .pro inhibitors, we used transfer learning to fine-tune a classification model for bioactivity prediction. The performance of the classifier varied depending on the split method used. For random splitting, the model achieved a validation AUC-PR of 0.310 ± 0.036 and a test AUC-PR of 0.220 ± 0.027. The performance using scaffold split was worse, with validation AUC-PR of 0.251 ± 0.083 and test AUC-PR of 0.185 ± 0.13 . Our model clearly outperformed the baseline for random predictions, achieving an AUC-PR of 0.255  at the cost of low recall (< 0.10). In addition, the probabilities output by the classifier were extremely low, with a median of 0.0035. The low probabilities are the result of the extreme class unbalance on the training set, with only 0.1% of active molecules. In future work, we will prioritize calibrating the probabilities to a more reasonable range and improve the recall. The model will be retrained as soon as more activity data is available for SARS-CoV-2 Mpro inhibitors in chemical space . Benzotriazoles similar to LaBECFar-1–4, have been described as non-covalent inhibitors of SARS-CoV-1 Mpro and a X-ray crystal structure between a prototype bound to the enzyme is available on the Protein Databank (PDB: 4MDS) [LaBECFar-5–10, have been described as covalent inhibitors of SARS-CoV-1 Mpro [LaBECFar-9 is present on the training set and was rediscovered by our approach. The core peptidomimetic structure is preserved in the generated molecules and they also bear the warhead group benzothiazolyl ketone at P1′ position, which could form a covalent bond with the cysteine of the catalytic dyad Cys-His on the active site of Mpro.We also report the top-20 predicted molecules for Mion Fig. . These 2B: 4MDS) . PeptidoV-1 Mpro . In factpro in vitro [pro is available on PDB (PDB: 6WNP). Some of the top 10 molecules on Additional file pro, such as Novobiocin [We also applied our classifier to a list of 1959 FDA approved drugs to explore its potential for drug repurposing. Additional file in vitro –60. In avobiocin , Saquinavobiocin , 63, Aprvobiocin  and Leucvobiocin . Neverthpro (PDB: 6W79). Nine molecules were considered hits, displaying similar binding poses to experimentally validated inhibitors in X-ray crystal complexes with Mpro. These hits included three benzotriazoles  and four benzothiazolyl ketone , one peptidomimetic (LaBECFar-11), and one N-(2-pyridyl)acetamide derivative (LaBECFar-17).In order to further prioritize molecules for biological testing, we submitted the top-20 predicted hits to a docking simulation using the crystal structure of SARS-CoV-2 MLaBECFar-11 fits nicely into the active site of Mpro, showing a similar binding pose to peptidomimetic inhibitors described in other works, including 11a  developed by Turlington et al. [LaBECFar-4, the benzotriazole ring binds to the S1 pocket, formed by the side chains of F140, N142, H163, and H172 -acetamide group at position P1 in LaBECar-1 -acetamide group was accommodated between the S2/S1′ pockets and established an H-bond with G143 . The γ-lactam group at P1 position established H-bond with the imidazole of H163 and the side chain of E166 on the S1 subsite. The leucine side chain at P2 inserted into the S2 pocket and formed hydrophobic interactions with M49, D187 and Y54. The 4-methoxy-benzyl group at P3 interacted with the solvent-exposed S4, forming a H-bond with Q192.The benzothiazolyl ketones r-5 Fig. b, LaBECF-20 Fig. a and SARLaBECFar-17; bearing an acrylamide moiety that could covalently inhibit Mpro . The docked pose of LaBECFar-17 revealed that the 3,4-dichloro group inserts into the S2 pocket, while the pyridine ring forms a H-bond with H163 at S1  filter implemented in the FAF-Drugs4 server in order to identify molecules with the potential to interfere with biological assays. Not surprisingly, LaBECFar-15 and 16 were flagged as PAINS; the amino-thiophene group in these molecules is known to have thiol reactivity. The other 18 predicted hits passed in all PAINS filters.pro inhibitors often rely on warhead groups, such as α,β-unsaturated carbonyls, aldehydes and thiol-reactive esters [pro inhibitors and should not be discarded before experimental confirmation of bioactivity and that they are not interfering with the biological assay.In silico filtering using predefined rules is a valuable tool to prioritize molecules from huge databases and reduce the risks of false positives in biological assays , 70. Mane esters , 73; thee esters , 75. Thepro. The chemical space of the generated molecules overlapped with the target chemical space of Mpro inhibitors, showing that the key structural features were properly captured by the model. In addition, the generated molecules and real Mpro inhibitors showed similar physicochemical properties.We used ULMFit to train a chemical model for de novo design and fine-tune a classifier for bioactivity prediction on SARS-CoV-2 Mpro. The predicted active molecules also shared scaffolds with real Mpro inhibitors, while introducing a range of changes to lateral chains and the core of the scaffolds, indicating it could be used to explore the structure–activity of chemical series.The fine-tuned classifier outperformed the random classification baseline and a model that is being used to repurpose drugs for SARS-CoV-2 Mpro, we will update the model in order to generate more diverse and novel molecules.We also highlight that the current version of our generative model is still limited by the nature of the training data. As more molecules are screened against MAdditional file 1:Table S1. Validity, uniqueness and novelty (mean ± std) of SMILES generated after training. We sampled 10,000 SMILES for eachtemperature . Figure S1. UMAP plot of the chemical space of scaffolds generated by the general chemical modeland scaffolds from ChEMBL . Figure S2. Redocking experiment to validate the molecular dockingprotocol. The docked pose of ligand X77 from SARS-COV-2 Mpro (PDB: 6W79) is shown as purple sticks and the experimental binding pose as greensticks. The enzyme surface is shown in bege. The RMSD between the docked and experimental pose was 1.106 Å. Figure S3. Docked poses of LaBECFar-1and LaBECFar-3 on SARS-COV-2 Mpro. (PDB: 4MDS). The amino acid residues are shown as bege sticks and the ligands are shown as pink sticks.Figure S4. Docked poses of LaBECFar-6, LaBECFar-7 and LaBECFar-9 on SARS-COV-2 Mpro. (PDB: 6W79). The amido acid residues are shown asbege sticks and the ligands are shown as orange sticks. Table S2. FDA approved drugs predicted to be active on SARS-CoV-2 Mpro.


B9Drosophila melanogaster models of PD. We found that undifferentiated CAD cells were more susceptible to vanadium exposure than differentiated cells, and this susceptibility was modulated by iron. In PINK−1 flies, the exposure to chronic low doses of vanadium exacerbated the existing motor deficits, reduced survival, and increased the production of reactive oxygen species (ROS). Both Aloysia citrodora Paláu, a natural iron chelator, and Deferoxamine Mesylate (DFO), a synthetic iron chelator, significantly protected against the PD-like phenotypes in both models. These results favour the case for iron-chelation therapy as a viable option for the symptomatic treatment of PD.Parkinson’s disease (PD) pathology is characterised by distinct types of cellular defects, notably associated with oxidative damage and mitochondria dysfunction, leading to the selective loss of dopaminergic neurons in the brain’s substantia nigra pars compacta (SNpc). Exposure to some environmental toxicants and heavy metals has been associated with PD pathogenesis. Raised iron levels have also been consistently observed in the nigrostriatal pathway of PD cases. This study explored, for the first time, the effects of an exogenous environmental heavy metal (vanadium) and its interaction with iron, focusing on the subtoxic effects of these metals on PD-like oxidative stress phenotypes in Catecholaminergic a-differentiated (CAD) cells and PTEN-induced kinase 1 (PINK−1) Parkinson’s disease (PD) is the second-most prevalent neurodegenerative disease, after Alzheimer’s disease, and affects from one to five percent of the world’s elderly population, i.e., over 60 years to over 85 years, respectively . Its prehttps://clinicaltrials.gov/ct2/show/NCT02655315; https://clinicaltrials.gov/ct2/show/NCT02728843).Several heavy metals are required for vital physiological processes in the body, including the brain. Useful metals in the brain include copper, a vital cofactor for enzymes in neurotransmitter synthesis; zinc, which is needed in synaptic transmissions and regulates NMDA glutamate receptor activity; iron, for systemic oxygen transport, myelin, and neurotransmitter synthesis; and manganese, a cofactor in metalloproteins and associated with enzyme functions, including hydrolases and lyases ,7,8,9. I2O5) and, thus, is emitted by welding rods commonly used in construction.Vanadium (VD) is the fifth-most abundant transition metal in the Earth’s crust . It is kLow-dose VD is known to have some health benefits, notably blood sugar-lowering properties in diabetic patients and supplements in leukaemia treatments . HoweverIron and VD are both transition metals, which are known to cause oxidative stress by the generation of superoxide and hydroxyl radicals through the Fenton reaction. This leads to free radical-mediated damage to proteins, membranes, and DNA . VanadiuB9 model of PD. The interplay between VD and endogenous iron was, consequently, explored in both models.The aim of this study was to establish the effects of the exogenous environmental heavy metal VD on the neuronal mitochondrial viability and oxidative status, using a monoaminergic CAD (Cath. a-differentiated) cell line in vitro, and, thence, the motor activity, brain oxidative status, and life span, using the in vivo PINK-1The first set of experiments were designed to explore the dose-dependent effects of vanadium (VD) on a range of immature and mature monoamine neurons in the culture. Acute (one-day) and chronic (five-day) treatments with graduated doses (10–1000 μM) of sodium metavanadate (vanadium) were performed for undifferentiated and differentiated cells. The mitochondrial viability investigation revealed that undifferentiated cells were more sensitive to the toxic effects of both acute (from 100 μM) and chronic (>20 μM) administrations of VD, but differentiated cells were only affected by chronic administration (from 100 μM) A. Both dp < 0.005) levels of intracellular iron (about a three-fold increase) in undifferentiated versus differentiated CAD cells  upon vanadium (200 μM)-induced oxidative stress  following chronic (six-day) exposure in differentiating CAD cells was investigated. Both DFO and A. citrodora oil significantly reversed vanadium-induced toxicity, as compared with vanadium-only treated cells (The effects of both synthetic (DFO) and natural iron chelators (ed cells .p > 0.05); however, a modest increase in motor activity was seen in flies treated with L-dopa (* p < 0.05) compared to the control group (p < 0.01), while L-dopa ameliorated them  was observed (p = 0.0349) relative to the control group was observed, with the median survival for the VD-treated group being five days compared to eight days for control flies. There was no significant effect on survival in the L-dopa-treated group relative to the control, with the median survival for both groups being eight days . However, a significant increase in the survival of flies treated with L-dopa (** observed A. In PINght days B.p < 0.0001) compared to VD alone and to control PINK-1 mutant flies  + VD (**** nt flies A. Concomnt flies B. Convernt flies .p < 0.05) but significantly reduced the T-SH levels in PINK-1 mutant flies (* p <0.05), compared to the control and VD-treated PINK-1 mutant flies  levels in the wild-type and PINK-1 mutant control flies, we observed a surprisingly high T-SH baseline level in the mutant compared to the wild-type flies. Chronic low-dose VD (1μM) elicited a reduction in T-SH in WT flies but no significant effect upon PINK-1 mutant flies. Iron chelation (DFO) reversed the effect of VD on TS-H levels in the WT flies to the control levels  toxicity and Parkinson’s disease (PD). The ability of VD to generate reactive oxygen species (ROS) in a Fenton-like reaction has been reported , but thetial oil , complettial oil . Furthertial oil ,36.The PTEN-induced kinase 1 (PINK-1) gene encodes a kinase located on the membrane of the mitochondrion , and theThe next stage of the study explored the effects of a subtoxic concentration of VD and the role of iron upon wild-type and PINK-1 mutant loss-of-function flies as a model of familial Parkinson’s disease with a mitochondrial respiratory exchange deficit. In wild-type flies, the dopamine precursor L-DOPA, which rescues dopaminergic neurones in cellular stress, elicited a significant increase in both the motor activity and survival, while vanadium had no significant effect (positive or negative).B9Drosophila model of PD. This is in contrast to our findings, but it should be noted that the flies in this present study were raised on a different genetic background. Furthermore, our study measured the total thiol levels rather than the GSH-T. In certain situations, there can be an elevated thiol level in response to acute oxidative stress, as seen in a report of 24-h exposure to paraquat in Drosophila flies [VD was shown to significantly reduce and increase ROS levels in WT and PINK-1 mutant flies, respectively. Thiols  are prominent components of the body’s antioxidant defence system that aid the protection of proteins from ongoing irreversible oxidative damage . The totla flies . Our stuLow-dose subtoxic VD administration in normal cells leads to a promotion of cellular wellbeing, possibly through the modulation of growth factor-mediated signal transduction pathways and the promotion of cell transformation . In cellTo explore the role of iron on the effects of VD, the iron chelator DFO was employed again. Coadministration of the DFO with VD in WT flies restored the reduced ROS and thiols levels caused by VD alone back to the control levels . PINK-1 Both iron and VD are known to bind to transferrin in the circulation , but theA. citrodora are known iron chelators that mop up excess iron in the system, both in vivo and in vitro. However, no chelator is entirely specific for one given metal; both DFO and deferiprone are known to chelate metals other than iron (3+) [+) in vivo [The function of iron chelators in mopping up excess iron ions would make them useful in mitigating against effects observed from the “iron-mediated oxidative damage” caused by VD administration, as observed in this study. This is further illustrated by , who repron (3+) . In fact in vivo ,57,58. T in vivo  reported in vivo , we can The monoaminergic CAD (Cath. a-differentiated) cell line  exhibit biochemical and morphological characteristics of mature differentiated primary neurons, expressed enzymatically active tyrosine hydroxylase, and accumulated L-dopa, the precursor of dopamine.Aloysia citrodora Paláu essential oil, determined by gas chromatography-mass spectrometry (GC-MS), as described previously , was utilised for this study.Chemically characterised 2 on 75-cm2 tissue culture flasks  in Dulbecco’s modified Eagle’s medium DMEM⁄F-12 Media-GlutaMAX™-I , supplemented with 10% foetal bovine serum . Cells were passaged every 3 to 4 days at a 1:4 dilution. To differentiate CAD cells, the growth media (with 10% FBS) in which the cells had been plated was removed gently and replaced with serum-free DMEM Nutrient Mixture F-12. After replacement of the serum-free media, the cells were returned to the incubator and left for six days to differentiate (DIV6). Differentiation was confirmed by microscopic analysis and MAP2 immunolabelling (not shown).CAD cells were grown at 37 °C and in 5% COTo determine the cell mitochondrial viability, the MTT  assay was performed as previously described ,61. The 2 on 75-cm2 tissue culture flasks  in Dulbecco’s modified Eagle’s medium DMEM⁄F-12 Media-GlutaMAX™-I , supplemented with 10% FBS. CAD cells were passaged and seeded as previously described into two 75-mL flasks. After 24 h, the process of differentiation was initiated. After 100% confluency was achieved, the undifferentiated cells were rinsed with cold PBS and detached. After detaching, the falcon tubes were centrifuged for 5′  and transferred into 1.5-mL Eppendorf tubes, re-centrifuged, and stored in −20 °C for Inductively coupled plasma mass spectrometry (ICP-MS) analysis for metal contents. Similarly, the differentiated (6 days DIV) cells were detached and stored in −20 °C for ICP-MS.CAD cell cultures and experiments were performed in Containment Level 2 facilities under sterile conditions. CAD cells were grown at 37 °C and in 5% CO3, 65%) was added to each thawed cell pellet and vortexed. The cells were left for 24 h to be digested in the fume hood and vortex-mixed again. One point two millilitres of digested sample was removed and dispensed into fresh Eppendorf tubes and centrifuged . Five hundred microlitres of a digest was then diluted with 4.5-mL 2.5% HNO3. One millilitre of the clarified sample was decanted into a 15-mL falcon tube. The clarified sample was diluted 1/10 (2% HNO3). The analysis was carried out with Ag as an internal standard (Ag 100 ppb), which was added to the samples and standard curve. An ICP-MS analysis was performed in duplicates.One point five millilitres of nitric acid , and these were used to define the limits for quantification. The final working range was used to quantify the metal content.Drosophila melanogaster wild-type Dahomey (WT) and PINK-1 (w-PINK-1 B9/FM7.GFPw+) mutant flies were used for this experiment. Flies were maintained in an incubator with a 12-h day-night cycle at 25 °C. Fly food was prepared by mixing 15.025 g of instant medium  with 46 mL of deionized water (formula with this food: water ratio was found to have the best consistency) per bottle . Following this, each bottle was shaken to ensure even distribution of the water—after which, a few grains of baker’s yeast were added to each bottle. Each bottle was plugged with a foam plug and left to set for at least an hour at room temperature before transferring the flies (progenies) into them. Fresh food was prepared every two weeks, and flies were flipped into new bottles every 2 to 3 days.n = 10 per bottle per group). Treatments were commenced from the larval stage and continued throughout the experiments. The groups  tested comprised: a control group (with no treatment), positive control (treated with L-dopa 13.4 µM), a treatment group , and another treatment group (VD + DFO (10 µM)) to investigate the influence of iron chelation. To prevent the oxidation of L-Dopa, 1.6 µM of ascorbic acid was added to each bottle containing L-Dopa [This study explored the effects of chronic exposure of VD on the motor activity, lifespan, and oxidative stress markers .At the end of exposure to vanadium, the flies from each group of control and treated (vanadium only and vanadium + DFO) groups were anaesthetized on ice, snapped frozen in liquid nitrogen, and vortexed at high speed to separate the fly head from the body. The detached fly heads were transferred into pre-weighed Eppendorf tubes and weighed. They were then homogenised in 0.1-M phosphate buffer, pH 7.0 (1 mg: 10 µL), centrifuged for 10 min at 4000 g (4 °C). The supernatants obtained were stored at −20 °C and used to determine the ROS level, total thiol, and protein content. The assays were performed in duplicates (n = 3 replicates. The rate of DCF formation was expressed in percentage of the control group.To determine the ROS level following chronic exposure to vanadium and the influence of Fe chelation and 2′ and 7′-Dichlorofluorescein (DCFH), oxidation was measured as an index of oxidative stress using a 96-well plate . The fluThe total thiol content for both WT and mutant flies was determined . The rea


In the original article, there was a mistake in the legends for Figures 2 and 3 as published. The ratings should be “(from 0 to 3)” and not “(from −5 to 5)”. The corrected figure legends appear below:Figure 2. Results of the Acquisition phase. Skin conductance responses (SCRs) (peak-trough) were z-scored, log-transformed, and averaged across participants in each group. Unconditioned stimuli (US) expectancy ratings (from 0 to 3) were averagxed across participants in each group. Overall, participants in both the 2-methyl-2-thiazoline (2MT) and Control groups were similarly successful in fear acquisition. For both SCR and US expectancy, mean responses to the conditioned stimuli (CS)– were subtracted from those to the CS+ s to display fear-specific effects. Error bars represent standard error of the mean. (A) The SCRs of both groups, were significantly greater to the two CS+ s than to the CS–. This provides evidence for successful acquisition of CS–US associations. (B) Likewise, the US expectancy ratings of both groups, were significantly greater to the two CS+ s than to the CS.”“Figure 3. Results of the Test phase. Skin conductance responses (SCRs) (peak–trough) were z-scored, log-transformed, and averaged across participants in each group. Unconditioned stimuli (US) expectancy ratings (from 0 to 3) were averaged across participants in each group. Mean responses to the conditioned stimuli (CS)– were subtracted from those to the CS+ s to display fear-specific effects. Furthermore, because no effects of the odor context used in the Test phase were found, mean responses were averaged across these for ease of display. Error bars represent standard error of the mean. (A) Overall, for the 2-methyl-2-thiazoline (2MT) group, SCRs were significantly stronger to the two CS+ s than to the CS–. No significant differences were found for the Control group. For demonstrative purposes, independent-sample t-tests were conducted to compare (CS+ – CS–) between groups (collapsed across the two Odor Contexts used in the Test phase). Both showed significant differences (**p < 0.01). (B) On average, both groups rated US expectancy as higher on the CS+ than on the CS– trials, with no apparent difference between the groups.”“In addition, the Supplementary Figures are missing legends. The correct legends appear below.Supplementary Figure 1. Odor-matching in main experiment . Overall, there were no significant differences in odor ratings between the 2MT and Control groups. Error bars represent standard error of the mean. These results suggest that any between-group fear memory differences found in this experiment cannot be explained by how pleasant, familiar, and/or how liked the different odors were.”“Supplementary Figure 2. Detailed SCR results of the Test phase of the main experiment. SCRs (peak-trough) are z-scored, log-transformed, and averaged across participants in each group. Overall, SCRs of the 2MT group were greater to the two CS+s than to the CS– in both odor contexts. No such difference was found for the Control group. For demonstrative purposes, for each odor context, paired-sample t-tests were conducted within each group to compare SCRs to the different CS . Error bars represent standard error of the mean.”“Supplementary Figure 3. Odor-matching in Follow-Up Cortisol Experiment . Overall, there were no significant differences in odor ratings between the 2MT and Control groups. Error bars represent standard error of the mean.”“Supplementary Figure 4. Detailed results of the follow-up Long-Term Test. SCRs (peak-trough) are z-scored, log-transformed, and averaged across participants in each group. Unlike in the analysis for the results of the original Test, analysis of SCRs in the follow-up Long-Term Test showed no significant effect of CS. Therefore, in this figure, mean responses to the CS– were not subtracted from those to the CS+s. US expectancy ratings (from 0 to 3) were averaged across participants in each group. Error bars represent standard error of the mean. The top graph shows SCRs, which appear to be higher for the 2MT than for the Control group in both odor contexts. The bottom graph shows US expectancy ratings, which also appear to be higher for the 2MT than for the Control group, except for those to the CS- in the Extinction Odor Context.”“Supplementary Figure 5. Results of the Extinction phase. SCRs (peak-trough) are z-scored, log-transformed, and averaged across participants in each group. Mean responses to the CS– were subtracted from those to the CS+s to display fear-specific effects. Error bars represent standard error of the mean. Overall, neither group showed significant differences between SCRs to the Extinction CS+ and the CS–. This indicates that there was a lack of fear-like responses to the Extinction CS+ during the Extinction phase.”“Finally, a file is missing in the Supplementary Material. It has now been published as Data Sheet 1.The authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated.